Process of producing l-glutamic acid by fermentation



United States Patent PROCESS OF PRODUCING L-GLUTAMIC ACID BYFERMENTATION Akira Kamimura, Kawasaki-shi, and Ryuicbiro Tsugawa andShinji Okumura, Tokyo, Japan, assignors to Ajinomoto (30., Inc., andSanraku Ocean Co., Ltd., both of Tokyo, Japan No Drawing. Filed Feb. 20,1968, Ser. No. 706,774 Claims priority, application Japan, Feb. 28,1967,

Int. Cl. C12b 1/00 US. Cl. 195-30 5 Claims ABSTRACT OF THE DISCLOSUREThe yield of L-glutamic acid from an aqueous fermentation mediumcontaining acetate ions as the principal carbon source andmicroorganisms capable of producing glutamic acid from such a carbonsource may be increased significantly by the addition of an assimilablesulfur source to the medium. Suitable sources include cystine, cysteine,homocystine, homocysteine, methionine, thioglycolic acid,B-mercaptoethanol, and thiourea, and are employed at initialconcentrations of 0.005 to 2%, preferably 0.05 to 0.5%.

This invention relates to the production of L-glutamic acid by microbialfermentation, and particularly to the production of L-glutamic acid froma nutrient medium containing acetate ions as the principal source ofcarbon.

It is known that many microorganisms are capable of producing L-glutamicacid by aerobic fermentation of nutrient media containing acetate ionsas the principal carbon source (T. Tsunoda et al., J. Gen. Appl.Microbiol. 7 (1961) 18; Tanaka et al., U.S. Pat. No. 3,335,065 andFrench Pat. No. 1,424,809; U. A. Phillips, US. Pat. No. 3,277,625). Ascompared to procedures employing sugars as carbon sources, the rate ofglutamic acid formation from acetate ions is rather slow, and theultimate yields are significantly lower.

We have now found that the known process can be substantially improvedby adding to the nutrient medium employed a source of assimilable sulfurin an amount much greater than the sulfur traces provided by sulfurbearing amino acids and the like in such conventional nutrients asprotein hydrolyzates, yeast extract, or corn steep liquor. The compoundsmost effective in stimulating the production of glutamic acid fromacetate ions are the sulfur-bearing amino acids cystine, cysteine,methionine, homocystine, homocysteine, but substantial improvements arealso achieved with very different organic sulfur-bearing compounds, suchas thioglycolic acid, B-mercaptoethanol, and thiourea. Both opticallyactive L-forms and inactive DL-forms of the sulfur-bearing amino acidsmay be employed.

The lower limit of effectivness of the sulfur sources appears to dependentirely on the sensitivity of the analytical method employed fordetecting the effect. Practically significant results are obtained whenthe nutrient medium contains only 0.005% (by Weight) of the sulfursource. Greatest improvement is achieved at concentrations of 0.05 to0.5%. At higher concentrations, the cost of the sulfur source tends tooffset the higher glutamic acid yields, and at concentrations greaterthan 2%, the yields of glutamic acid again decrease. Measurableimprovement, however, can be detected at much higher concentrations. Theeffect of the sulfur-bearing compounds is cumulative within thepreferred ranges, so that several compounds may be employed jointly inmixtures.

An effective concentration of sulfur-bearing compounds cannot beachieved in the conventional nutrient media by merely increasing theingredients which normally provide trace amounts of assimilable sulfur,such as the protein hydrolyzates and other supplemental nutrientsreferred to above. It appears that the relatively large amounts of aminoacids free from sulfur which are introduced into the nutrient media bythe necessary amounts of protein hydrolyzates have an inhibiting effectwhich by far outweighs any benefits that could be obtained from theincreased sulfur supply.

The composition of the basic nutrient media prior to modificationaccording to this invention may be entirely conventional. They shouldcontain a source of assimilable carbon mainly constituted by acetateions, a source of assimilable nitrogen, organic growth promoters, andthe inorganic ions necessary for microbial growth. The acetate ions maybe supplied by alkali metal or ammonium salts of acetic acid or by thefree acid, When ammonium acetate is employed as a source of carbon, itsimultaneously supplies nitrogen.

All microorganisms known to produce L-glutamic acid from acetate ions asthe principal carbon source respond to the addition of sulfur-bearingcompounds according to this invention in substantially the same manner.As will partly be illustrated hereinbelow, all tested strains ofMicrococcus, Corynebacterium, Brevibacterium, and Arthrobacter whichproduce L-glutamic acid from acetate ions in the absence of the sulfurbearing compounds of the invention produce glutamic acid at a fasterrate and to higher ultimate concentrations when the sulfur-bearingcompounds are present. These benefits are not lost if the acetate ionsare replaced in part with sugars as a carbon source.

It is normally preferred to operate at relatively low initial acetateion concentrations, and to replenish and increase the acetate content ofthe nutrient medium during the growth period of the microorganisms, theadditions starting approximately in the middle of the logarithmic phase,and to keep the pH between 7.0 and 8.5 by the added material.

The following examples are further illustrative of this invention, butit will be understood that the invention is not limited thereto.

' EXAMPLE 1 An aqueous nutrient medium was prepared to the followingcomposition:

Ammonium acetate: 2.0%

MgSO -7H O: 0.04%

Fe++, Mn++ (each): 2 ppm. Thiamine hydrochloride: 200 y/liter Biotin:2.0 'y/liter Casamino acid (DIFCO): 0.2%

The casamino acid employed was free from vitamins and contributed to themedium 91 g./ ml. of amino acids (total nitrogen 14 mg./ 100 ml.),including 0.026 mg./ 100 ml. L-cystine and 2.8 mg./ 100 ml.L-methionine.

Three 20 ml. batches (A, B, C) of the medium were sterilized in 500 ml.shaking flasks (Sakaguchi type) after 0.015% L-cystine had been added tobatches B and C, and additionally 0.005% L-methionine to batch C. Thethree media were inoculated with Brevibacterium lactofermentum No. 2256(ATCC 13869) which had been cultured on bouillon agar slants for 24hours, and shaken for 10- 24 hours at 31 C. The L-glutamic acid contentof batch A after 10 and 24 hours respectively was 2.5 and 3.7 mg./ ml.The corresponding values for batch B were 3.2 and 5.3 mg./ml., and forbatch C, 3.3 and 5.6 mg./ml. Addition of the sulfur bearing amino acidsraised the glutamic acid yield by approximately one half.

3 EXAMPLE 2 An aqueous nutrient medium was prepared to the followingcomposition:

Ammonium acetate: 2.0%

4 EXAMPLE Ammonium acetate: 1.0%

Glucosfil 5 Sodium acetate: 1.0% 2 Z 0.1% Glucose (starch hydrolyzate):1.5% 2 4 2 (104% Thiamine hydrochloride: 200 'y/ liter Fett, 2 arBiotin: 2 'y/liter Thiamine hydrochloride: 200 /liter 10 1 1 1 Biotm:2.0 v/l ter O 1% Soybean protein hydrolyzate (A 1-ek1): 1.0 ml./dl. s q0 P FeSo -7 H O: 0.001%

The Aji-eki, a commercial product, contributed 0.9 M11504: 0-O01%mg./100 ml. L-cystine and 0.8 mg./100 ml. L-methionine. Urear Threebatches of this medium respectively containing y f 9 no additionalcystine and methionine, added cystine, and DL-methlonme: 003% addedcystine methionin? 5 inoculated with the Three liters of the medium wereplaced in a 5-liter jar same mlcroqgamsm as descnbed m Example 1 and thefermenter, sterilized, and inoculated with Brevibacterium culture medlawere P F for L'glutamlc acld content lactofermentum as in Example 1. Themedium was then after bolus of l l q L- agitated under aerobicconditions at 31 C. for 24 hours. glutam c acid per milliliter of themedium were found when the microorganisms had grown to Produce signifirespectlvely' E LE cant amounts of L-glutamic acid, a solutioncontaining XAMP 3 25 equal amounts of acetic acid and ammonium acetatewas Eight flasks containing the culture medium of Example continuouslyfed to the medium at a rate to keep the pH 1 and varying amounts ofcystine were inoculated and between 7.8 and 8.0. The total amount ofacetate ions cultured aerobically as described in Example 1. The L addedover a cultivation period of 39 hours was 12.31 glutamic acid content ofeach medium was determined g./dl., and the glutamic acid concentrationreached 7.35 after 15 hours with the following results: g./dl.

Added L cystine (mg./dl.) 0 3 5 15 15 500 1,000 2, 000 L-glutamic acid(mg/m1.) 2.6 3.0 3.1 3.4 3.7 3.9 3.8 3.4

Significant improvement in the glutamic acid yield is If it is assumedthat the glucose present was converted thus achieved at all testedconcentrations of cystine, the to glutamic acid at a yield of 50%, theyield from the yield exceeding 3.0 mg./ml. from 0.005% to 2% cystineacetate was 53.6%. 165 g./crude L-glutamic acid crystals in the nutrientmedium. From an economic point of view, were recovered from the mediumin a conventional manthe most desirable results are achieved at cystineconcenner by removing the microbial cells and adjusting the pH trationsof 0.05 0.5% (5-500 mg./dl.), as discussed 40 of the remaining liquid to3.2. above. When the same procedure was repeated with a medium EXAMPLE 4free from added cystine and methionine under otherwise The eightcompounds listed in Table 1 which are igegtcal conditions, the yieldbased on acetate Was only sources of assimilable sulfur for themicroorganisms em- EXAMPLE 6 ployed, were added in the concentrationsindicated to respective batches of a nutrient medium which differed fromThe procedure of Example 5 was repeated with minor that described inExample 1 by slightly lower contents of changes with othermicroorganisms known to produce biotin (1.5 'y/ liter) and Casamino acid(0.1% and the glutamic acid by fermentation on acetate media. Twonuseveral batches were inoculated and cultured as described trient mediawere employed with each microorganism, in that example. The glutamicacid concentration in each Medium A containing 0.05% L-cystine inaddition to the batch and in a blank was determined after 24 hours, andingfediflnts Of d u B Wh h had the following the results obtained arelisted in the table as the percentcomposition; age y1eld, based on theacetate originally present. Ammonium acetate; 10%

Sodium acetate: 1.0% Ammonium sulfate: 0.5%

KH PO 0.1% TABLE 1 4' 2 074% Concem Fe++, Mn++ (each): 2 p.p.m. tuition,Thiamine-H01: 200 'y/liter Percent Yield Biotin: 1.5 'y/liter Sulfursource: Casamino acid (DIFCO): 0.2%

ilfsirasi::::::::::3::::::::::::::::::::::::: "005' 5333 giijg gg n 8 5Table 2 lists for each strain tested the concentration of g g mL-glutamic acid (GA), the" yield in percent of the acetate 411,6 3 ionsupp ied, and acetic acid or acetate (Ac.ac.)confiifltififitiitltfitfiff; j 8 8; sumed over a cultivation period of40 hours in Media A Thioul'la Q02 and B. While the several strains ofmicroorganisms differ greatly from each other in their ability ofaccumulating L-glutamic acid in the media used, the ratio of ultimateglutamic acid concentration between Medium A and Medium B is practicallythe same for all microorganisms, and As is evident from Table 1, theyield-improving effect the eifect of the sulfur-bearing addition agentsof the inof added assimilable sulfur sources is not limited to sulfurvention is unrelated to the specific nature of the employed bearingamino acids although the amino acids generally microorganisms capable ofconverting acetate ion t are more effective than other sulfur sources. 75 glutamic acid.

TABLE 2 Medium A Medium B GA Yield, Ae.ac. GA Yield, Ac.ao.Microorganism g./dl. percent g./dl. g./dl. percent g./dl F Bret]. flavumNo. 2247 (ATCC 14067). 4. 81 46. 10. 46 2. 89 34. 9 8. 29 v! Brev.roseum N0. 7 ATCC 13825 5.02 49.1 10.23 2.97 35. 1 8.47 5 Brev.Saccharolyticum No. 7636 5 (ATCC 14066) 3.54 p 45. 9 7.72 2.12 31.3 6.78Micrococcus glutamicus No. 541 T (ATOC 13058) 3.75 44.5 8.43 2.63 33.87, 79 Cory. acetoacidophzlu'm No (AT 870) 3. 80 44. 4 8. 55 2. 29 29. 27. 85 C011]. lilium (NRRL 224 3.90 42. 7 9.13 2.38 28.9 8.24 Cory.herculis (ATCC 13868) .17 42.5 9.81 2.59 29.9 8.66 Arthrobacter citreus23-2A (ATCC 1 While the invention has been described with particularcysteine, homocystine, homocysteine, methionine, thioreference tospecific embodiments, it is to be understood that it is not limitedthereto, but is to be construed broadly and restricted solely by thescope of the appended claims.

What is claimed is:

1. In a process of producing L-glutamic acid by aerobically culturingmicroorganism capable of utilizing acetate ions as a carbon source inthe fermentative production of said glutamic acid on a nutrient mediumcontaining said' acetate ions as the principal source of assimilablecarbon, the improvement which comprises:

(a) adding to said medium an amount of an assimilable source of sulfurefiEective to increase the yield of said glutamic acid.

2. In a process as set forth in claim 1, said source of sulfur beingadded to said medium in an amount of 0.005 to 2 percent.

3. In a process as set forth in claim 2, said source of sulfur being amember of the group consisting of cystine,

glycolic acid, fi-mercaptoethanol, thiourea, and mixtures thereof. i Y

4. In a process as set forth in claim 3, said source of sulfur being anamino acid.

5. In a .process as set forth in claim 4, said source of sulfur beirigadded to said medium in an amount of 0.05 to 0.5 percent.

References Cited UNITED STATES PATENTS 2,953,499 9/ 1960 Katagiri et a1.-47 3,117,915 1/1964 Shiio et al. 1'95-30 3,335,065 8/1967 Tanaka et a1.195-30 OTHER REFERENCES Chemical Abstracts, vol. 64, No. 15038a, b,1964.

A. LOUIS MONACELL, Primary Examiner G. M. NATH, Assistant Examiner

